A detailed study on genetic diversity, antioxidant machinery, and expression profile of drought-responsive genes in rice genotypes exposed to artificial osmotic stress

Seasonal variations in rainfall patterns, particularly during sowing, early growing season, and flowering, drastically affect rice production in northeastern India. However, sensitivity to drought stress is genotype-specific. Since 80% of the land in this region is used for rice production, it is crucial to understand how they have adapted to water stress. This study evaluated 112 rice genotypes grown in NE India for seed germination percentage and seedling development under PEG-mediated drought stress. Among the rice genotype, Sahbhagi dhan, RCPL-1-82, Bhalum-3 and RCPL-1-128 showed drought-tolerant traits, while Ketaki Joha, Chakhao, Chandan, RCPL-1-185 and IR-64 were the most drought-sensitive rice genotypes. Drought-tolerant rice also showed significantly higher seed germination potential, proline content, antioxidant activity and expression of drought-responsive genes than drought-sensitive rice genotypes. A similar expression pattern of genes was also observed in the rice genotype treated with a 50% water deficit in pot culture. In addition, drought stress reduced the pollen fertility and yield per plant in sensitive rice genotypes. Molecular markers associated with drought stress were also used to characterize genetic diversity among the rice genotypes studied.


Diversity analysis of rice genotype using SSR and RAPD marker
Preliminary genetic relationship among 112 studied rice genotype was assessed with 57 previously reported drought stress tolerance linked Simple Sequence Repeat (SSR) and 5 Random Amplified Polymorphic DNA (RAPD) markers.A total of 1395 alleles were scored using SSR and RAPD marker.Out of which 219 (15.7%) were monomorphic and 1176 (84.3%) were polymorphic.A representing banding pattern of studied rice genotypes were shown in Fig. 2. A summary of the molecular markers used in the present study were shown in the www.nature.com/scientificreports/Supplementary Table 2.In RAPD, OPB-10 showed highest range of allele size; while in SSR marker, RM3 shows more polymorphism and maximum number of alleles as compared to other primer and RM11 generated least number of alleles (Fig. 2).RM3233 showed highest range of allele size (165-269 bp).The allele frequency ranged from 0.25 to 0.71 with an average value of 0.46.The polymorphism information content (PIC) ranged from 0.48 to 0.93 averaging 0.77 (Supplementary Table 2).While RM3 showed the highest PIC value, RM204 showed the lowest PIC value, which indicates the potential use of these markers in genotyping and diversity analysis study.The model-based population structure analysis performed with STRU CTU RE software revealed highest K value of 3 (Fig. 2).A phylogenetic tree generated using NTSYSpc 2.10 based on similarity index SM coefficient divided the studied rice genotype into two clades (Supplementary Fig. 1).While clade-I is further dived into three clusters.Within Cluster-I, Sahbhagi Dhan, Bhalum-3, RCPL-1-82, Banglami and V-Dhan which showed high seed germination percentage and RWC formed one sub-group; In cluster-II, Full badam, Ketaki joha and RCPL-1-185 which also showed high RWC and seed germination % formed one group.On the other hand, Clade-II includes RCPL-1-128, chandan and IR-64 which showed drought-sensitive characteristics.The population of the 112 investigated rice genotype was found to be genetically diverse from each other.
Based on the physio-morphological traits and genetic diversity analysis, we further selected four rice genotypes showing two contrasting characters i.e., putative drought-tolerant (Sahbhagi Dhan and RCPL-1-82) and putative drought-sensitive (RCPL-1-185 and IR-64) for antioxidant profiling and molecular profiling of droughtsensitive genes.

Effect of PEG-stress on proline, H 2 O 2 , AaS, DHA and total ascorbate content in drought-tolerant/sensitive rice
As shown in Table 2, drought-tolerant and sensitive rice genotype showed significant differences in the accumulation of proline, H 2 O 2 , AaS content, DHA content and total ascorbate in shoots as well as roots on 15th days after PEG treatment.While accumulation of proline and H 2 O 2 in the roots and shoot of Sahbhagi Dhan and RCPL-1-82 enhanced with increase in PEG concentration, the same showed highest accumulation at 10% PEG treatment in roots and shoots of RCPL-1-185 and IR-64, thereafter declined with further increase in PEG concentration.Sahbhagi Dhan and RCPL-1-82 showed highest abundance of proline at 30% PEG treatment, while IR-64 and RCPL-1-185 showed highest proline content at 10% PEG treatment.Similarly, Sahbhagi Dhan www.nature.com/scientificreports/and RCPL-1-82 also showed significantly higher accumulation of H 2 O 2 as compared to RCPL-1-185 and IR-64.
Unlike proline and H 2 O 2 content, while AaS content declined with increasing PEG concentration, DHA content and total ascorbate increased in the roots and shoot tissues of all studied rice genotype at 0% to 30% PEG treatment.However, there were significant differences between the drought-tolerant (Sahbhagi Dhan and RCPL-1-82) and drought-sensitive (RCPL-1-185 and IR-64) rice genotypes.

Antioxidant enzyme activity in drought-tolerant and sensitive rice under PEG-stress
To decipher the comparative estimation of redox potential of antioxidants between drought-tolerant and sensitive rice genotypes, we performed antioxidant enzyme activity using six different enzyme assays viz.MDHAR, DHAR, GPX, GR, SOD and CAT activity assay.As shown in Fig. 3, antioxidant enzyme activity assays of MDHAR, DHAR, GPX and GR revealed a similar pattern of enzyme activity in the rice genotype treated with different concentration of PEG.In all four assays, enzyme activity was found higher at 10% PEG treatment as compared to control.However, enzyme activity declined with further increase in PEG concentration (10% to 30%) in the root and shoots of all four rice genotype studied.The rate of declining enzyme activity in drought-sensitive rice genotypes (RCPL-1-185 and IR-64) was much higher as compared to drought-tolerant rice genotype (Sahbhagi Dhan and RCPL-1-82).Whereas SOD activity increased with increase in PEG concentration in the root and shoot of all four rice genotype, CAT activity decreased from control to 30% PEG treated plants.While shoot showed significantly higher SOD and MDHAR activity, root showed significantly higher DHAR, GR, CAT and GPX activity.Additionally, Sahbhagi Dhan and RCPL-1-82 showed significantly much higher antioxidant activity in both roots and shoots in all PEG treatments as compared to RCPL-1-185 and IR-64.

Identification and expression pattern of drought-responsive genes in drought-tolerant/sensitive rice genotypes under PEG-stress
To understand the molecular mechanism underlying drought sensitivity in drought-tolerant/sensitive rice, we first identified 12 candidate reference genes viz.DREB1, p5cs, rbcs, AAO1, SOD, WRKY11, WRKY114, SDR1, NAC9, ZFP252, ZFP182 and DRAP1 which are directly or indirectly linked to drought stress based on available literature.The expression profile of these genes showed a significant difference between the tolerant rice genotype and the sensitive rice genotype at different PEG concentrations (Fig. 4).Expressions of DREB1, p5cs, SOD and WRKY11 were significantly induced with increasing PEG treatment in the roots and shoots of Sahbhagi Dhan and RCPL-1-82.In contrast, expression of DREB1 and p5cs showed the highest at 10% and 20% PEG respectively in the shoot and roots of RCPL-1-185 and IR-64, thereafter declined in expression.While expression of SOD and WRKY11 increased with increasing PEG concentration in root and shoots of Sahbhagi Dhan and RCPL-1-82.However, it was induced weakly by the PEG-mediated drought stress in RCPL-1-185 and IR-64.Although there was no significant difference in expression of DREB1, p5cs, SOD and WRKY11 between RCPL-1-185 and IR-64, Table 2. Effect of PEG mediated drought stress on proline content (µmol g −1 dry weight), H 2 O 2 (µmol g −1 fresh weight), AaS content (nmol g −1 fresh weight), DHA content (nmol g −1 fresh weight) and total ascorbate (nmol g −1 fresh weight).Each value is representation of mean ± sd, N = 9 and different letters along the row are statically significant at p < 0.05. it is clearly visible that DREB1, p5cs SOD and WRKY11 expressed much higher in Sahbhagi Dhan and RCPL-1-82 as compared to RCPL-1-185 and IR-64.Similarly, while expression of WRKY114 decreased in the tolerant rice line, it was induced with increasing PEG stress in the sensitive rice line.On the other, we did not find a significant difference in the expression of SDR1 in both root and shoot tissues of treated and control plants.While the expression of NAC9, ZFP252, ZFP182, and DRAP1 was upregulated with increasing PEG-mediated drought stress in Sahbhagi Dhan and RCPL1-82, these genes showed downregulation of expression in RCPL-1-185 and IR-64.

Effect of drought stress on pollen fertility, rice yield and expression of drought-responsive gene under pot culture
Pollen fertility is an important aspect in screening potent drought-tolerant and sensitive genotypes as it directly impacts seed setting and grain yield.The microscopic analysis of pollens stained with Potassium Idode stain (I 2 -KI) revealed a significant reduction in the pollen fertility percentage in drought-stressed RCPL-1-185 and IR-64 than in control (Fig. 5).No significant differences were observed in Sahbhagi Dhan and RCPL-1-82.As shown in Supplementary Fig. 6, Sahbhagi Dhan and RCPL-1-82 showed round-shaped, dark-coloured pollen  in Sahbhagi Dhan and RCPL-1-82, in contrast, a > 75% decrease in filled grains per panicle was observed in RCPL-1-185 and IR-64 (Fig. 5).Further, to check whether the pollen fertility and grain yield correlate with the expression of previously identified drought-responsive genes between different rice genotypes, we checked transcript abundance of DREB1, LOC_Os12g04500, LOC_Os02g50970, LOC_Os12g26290, LOC_Os05g08480, MYB80 and WRKY114 genes in the young panicle (Fig. 7).The results revealed significant reduction in expression of LOC_Os12g04500, LOC_Os02g50970, LOC_Os12g26290 and MYB80 genes in treated RCPL-1-185 and IR-64 (drought-sensitive)

Discussion
Abiotic stresses like droughts drastically affect crop performance by impairing growth, development, and productivity.However, the most effective way to alleviate drought stress in plants is by breeding drought-tolerant cultivars.In the past few decades, rice breeders have developed many drought-tolerant rice genotypes to improve the plant's resilience under drought conditions 13 .However, the effectiveness of any transgenic/hybrid variety depends on the adaptability to a particular habitat or environment.The primary way to achieve such a goal is by screening germplasm in the field and as well as greenhouse conditions.Since multiple factors affect the performance of a crop in the field condition, therefore, a well-maintained in-house condition may be efficient in finding better experimental results.Although drought affects the life cycle of any plant; seed germination stages, seedling stages and reproductive stages are the most critical stages that determine the growth and yield respectively 14,15 .According to the Indian Meteorological Department of India (IMD), NE-India has received 31% less rainfall since 2018.NE-India has also been witnessing a series of weather anomalies, which have directly affected the major staple crop of this region i.e., rice.Therefore, in this study, 112 rice genotypes grown in NE-India were taken into consideration for a detailed analysis against PEG-mediated drought stress and further validated with the field/pot culture study.The primary goal of this study was to identify some potent drought-tolerant cultivars specific to NE-India and analyse of detailed mechanism underlying drought tolerance traits.We included two rice genotypes developed by the International Rice Research Institute (IRRI) namely Sahbhagi Dhan as a positive control (drought-tolerant released genotype) and IR-64 as a negative control (drought-sensitive genotype).Our preliminary study on the effect of PEG-mediated drought stress on seed germination and relative water content showed Sahbhagi Dhan, RCPL-1-82, Bhalum-3, RCPL-1-128, Baglami and Bhutmari as potent drought-tolerant rice genotype.The increasing PEG concentration did have much impact on germination and RWC on these plants as compared to Ketaki Joha, Chakhao, Chandan, RCPL-1-185 and IR-64.These variations in phenotypic and physiological characteristics could be attributed to genotype-specific stress tolerance mechanisms 15 .Evan's dye staining of the roots of the selected rice line confirmed the enhanced accumulation of antioxidants in the tolerant rice genotype as compared to the sensitive genotype.
Our study on molecular profiling using drought stress-linked markers for the studied genotypes showed a high degree of diversification.The identified allelic variants in the form of amplified product size (molecular weight) for each SSR and RAPD marker were documented to find out the allele mining set for the linked markers of the studied genotypes in relation to drought stress tolerance.Since SSR markers are abundant in the rice genome, co-dominance, and have a high polymorphism rate, they are widely utilised in rice genetics 16 .Although RAPD markers are less reproducible; however, RAPD technology still has several key benefits, including its suitability for work on anonymous genomes, applicability to small amounts of DNA, and low cost 17 .Therefore, we selected more SSR markers [18][19][20] and only a few RAPD markers 21,22 to check if they would produce some significant results.The result revealed that RAPD marker OPB-10 and SSR marker RM3, RM3233 were the best-suited markers to check genetic diversities under PEG-mediated drought stress in rice as these markers showed the highest range of polymorphisms and maximum number of alleles as compared to other primers.It was interesting to see that most of the rice genotypes that showed drought-tolerant/sensitivity using phenotyping and physiological screening also formed separate groups in the phylogenetic tree generated via NTSYSpc-based SSR and RAPD marker.These preliminary results indicate a possible genetic linkage between the genotypes.Since the markers used are linked to drought/water stress-related genes, the result could indicate that each group may share common alleles or haplotypes of the water stress-related genes involved.However, further research is needed to establish a proper linkage between the genotypes.Based on the cumulative results and their sensitivity to drought stress, studied rice genotypes can be categorized into three groups such as tolerant (Group I), moderately sensitive (Group II) and highly sensitive (Group III).While Group I includes Sahbhagi Dhan, RCPL-1-82, Bhalum-3, RCPL-1-128, Group III includes Ketaki Joha, Chakhao amubi, Chandan, RCPL-1-185 and IR-64 and group-II includes the rest of the studied genotype (Supplementary Table 5).Our results showed a higher drought-tolerance capacity of RCPL-1-82 apart from Sahbhagi Dhan and Baglami.Our results of sensitive rice are in conformity with the Sahoo et al. 23 .They reported Ketaki joha and Chandan as among the sensitive rice genotypes.However, we also found that IR-64 and RCPL-1-185 were more drought-sensitive than Ketaki joha and Chandan.We observed purple rice var.Chakhao amubi is moderately sensitive to drought stress 24,25 .However, further in-depth research underlying the molecular mechanism of drought tolerance/sensitivity in pigmented rice is still required.The high level of drought stress inhibited seed germination (%), shoot and root length and RWC.This response of growth inhibition could be attributed to low osmotic potential, decreased wall extensibility, and decreased cellular expansion 26 .In addition, while the proline content in Sahbhagi Dhan and RCPL-1-82 increased sharply with an increase in drought level via PEG stress or in pot culture, it declined in RCPL-1-185 and IR-64.Accumulation of proline under drought stress protects cells by balancing the osmotic potential of cytosol with that of vacuoles 27 ; therefore imbalance in proline content may have also affected the growth and sensitivity to drought stress between the studied plants.It was observed that rice varieties with improved resilience to abiotic stress have higher rates of proline biosynthesis 28 .Elevation in superoxide anion production has been reported to be associated with increased drought stress in many plants 29 .While we observed enhanced H 2 O 2 content in Sahbhagi Dhan and RCPL-1-82, the same declined in RCPL-1-185 and IR-64.This phenomenon could be attributed to a breakdown of H 2 O 2 into OH and OH − in RCPL-1-185 and IR-64 30 .
Drought stress induces plant defence mechanisms to protect themselves from the negative effects of oxidative stress.Plants with higher levels of induced antioxidants have better resistance and tolerance to oxidative stress 31 .ABA production also plays a significant role by enhancing its production during drought; salinity, chilling, and freezing stress 32 .High drought conditions also promoted the antioxidant enzyme activity viz.SOD, GR, MDHAR, DHAR, and CAT activity in the tolerant rice genotype (Sahbhagi Dhan and RCPL 1-82) as compared to the sensitive genotype (RCPL-1-185 and IR-64).Our results are in conformity with Sharma and Dubey 33  www.nature.com/scientificreports/Our sub-experiment of 50% water deficit on these four rice genotypes revealed that drought stress reduced the tiller numbers, and pollen fertility, which ultimately affected the seed setting and reduction in grain yield in sensitive genotype (RCPL-1-185 and IR64), while no significant differences were observed in tolerant rice genotype (Sahbhagi Dhan and RCPL-1-82).Our results are in concurrence with Rang et al. 35 , Praba and Thangaraj 36 .
Recently, a number of drought-linked genes and transcription factors have been characterized in different plants 37,38 .It is known that the DREB1 gene is hypersensitive to ABA signalling and a positive regulator of heat, salt, and drought tolerance in rice 39 .While P5CS is known to promote root growth and plays a significant role in plant drought resistance by enhancing proline biosynthesis 40,41 .To find out whether the proline accumulation and antioxidant enzymes activity is regulated by the expression of respective genes, we carried out expression profiles of DREB1, p5cs, SOD and WRKY genes.PEG-mediated osmotic stress also induced expressions of drought-tolerant genes and transcription factors such as DREB1, p5cs, SOD, and WRKY.The expression profile of these genes under PEG-mediated osmotic stress was correlated with drought stress under pot culture.These results established Sahbhagi Dhan and RCPL-1-82 potent drought-tolerant rice line as compared to other studied varieties.In higher plants, WRKY TFs are expressed during environmental stresses, seed germination, seed dormancy, and other growth-related events 42,43 .The WRKY proteins also play key functions in immune response mechanisms during mechanical damage and wounding 44 and stress like drought stress 45 , heat shock stress 46 etc.According to Wu et al. 47 , overexpression of WRKY11 enhanced temperature and drought tolerance in the transgenic rice genotype.We have also found that osmotic stress enhanced the expression of WRKY11 in the tolerant rice line; on the contrary, the expression of WRKY114 was induced due to enhanced osmotic stress in the sensitive line.This indicates the possible role of WRKY114 as a negative regulation of drought stress in rice.Bo et al. 48and Song et al. 49 have also reported overexpression of Maize WRKY114, a GA-responsive gene that negatively regulated salt and drought tolerance in transgenic rice.OsWRKY114 limits rice stomatal closure, which negatively impacts the plant's ability to withstand drought 48 .Apart from these transcription factors expression of SOD, CAT, and GPX also determines the accumulation of respective antioxidants that enhances defence mechanisms against ROS 45 .On the other hand, there was no noticeable difference in the expression of SDR1.While NAC9, ZFP252, ZFP182, and DRAP1 expression increased in Sahbhagi Dhan and RCPL-1-82 with increasing PEG-mediated drought stress, their expression decreased in RCPL-1-185 and IR-64.Hu et al. 50eported the role of OsNAC9 in increasing grain yield and enhancing root diameter in drought-tolerant rice lines.Similarly, overexpression of ZFP252 and ZFP182 also conferred multiple stress tolerance such as drought and salt tolerance in transgenic rice 51 .OsDRAP1, a gene similar to DREB2, is also reported to confer drought tolerance in rice by Huang et al. 52 .Our results also support the involvement of DEGs in drought tolerance or sensitivity during flowering stages in rice 53,54 .In the current experiment of 50% water deficit under pot culture, the expression of LOC_Os12g04500 (response regulator receiver domain-containing protein) and LOC_Os12g26290 (alpha-DOX2), which are also known as the core of the jasmonic acid (JA) signalling pathway, was significantly increased under a prolonged drought period.Additionally, it has been shown that JA signalling genes function at key stages of drought stress 55 .Five genes, DREB1, LOC_Os12g04500, LOC_Os02g50970, LOC_Os12g26290, and MYB80, were found to have upregulated expression in drought-tolerant genotypes (Sahbhagi dhan and RCPL-1-82) and downregulated expression in drought-sensitive genotypes (IR64 and RCPL-1-185), indicating a positive relationship between these genes and drought tolerance.Pan et al. 56 reported that MYB80 plays a key role in regulating anther development and enhancing pollen fertility.In sensitive rice genotypes, the genes WRKY114 and LOC_Os05g08480 (cytokinin-O-glucosyltransferase-1) showed upregulation; however, in the tolerant rice genotypes, the same genes showed downregulation.Similar results were also reported by Ahmad et al. 57 in the young panicle of sensitive/tolerant rice genotypes.

Conclusion
Our experiment on the effect of artificial osmotic stress via PEG-mediated and soil/pot culture stress established Sahbhagi Dhan and RCPL-1-82 as potent drought-tolerant rice genotypes as compared to other studied genotypes.We found multiple factors involved in conferring drought tolerance in rice at both metabolite and molecular levels.This study formed the preliminary base for selecting drought-tolerant rice collected from North-East India.The characterized rice genotypes may further be utilized for genome editing or rice breeding programs.

Plant material and PEG treatment
Healthy seeds of 112 rice genotypes (Supplementary Table 4) were collected from different locations in North-East India in the year 2015-2016.Pure genotypes were grown and maintained in the greenhouse of the Department of Biotechnology, Division of Crop Science, ICAR Research Complex for NEH region, Umiam, Meghalaya, India.
In this experiment, these rice genotypes including Sahbhagi Dhan (drought-tolerant control) and IR-64 (drought-sensitive control) were subjected to osmotic stress.Healthy grains of each genotype were germinated on the individual Petri dishes.To induce osmotic stress, PEG-6000 (Merck-Schuchardt, Hohenbrunn, Germany) was used.The impact of PEG-induced osmotic stress was investigated using four treatments-T0 = 0% PEG (control condition), T1 = 10% PEG (− 0.19 MPa), T2 = 20% PEG (− 0.69 MPa), and T3 = 30% PEG (− 1.12 MPa) and applied in accordance with a completely randomised design 58 .The seed germination test was measured on the 7th DAT (Day After Treatment).After 1 week, seedlings with synchronized leaf emergence, healthy roots, and shoots were hydroponically grown (5 × Yoshida medium; diluted from 10 × Yoshida medium) in individual trays under the same PEG treatment for an additional 8 days 58 .Ten replicates were maintained for each genotype per treatment.We evaluated the water potentials prior to and following the addition of PEG to determine the osmotic stability of the nutrient solutions.Individual plant of each genotype under each treatment was taken into www.nature.com/scientificreports/account for the physio-molecular analysis of root and shoot tissues, making each plant a separate experimental unit.pH was adjusted at 6.0 ± 0. 2 °C.Antioxidant activity and gene expression profiles on root and leaf tissue were conducted on the 15th DAT.
To check the expression profile of genes under PEG-mediated drought stress correlates with field experiments, a separate experiment was conducted on pot culture with a 50% water deficit 59,60 for four genotypes.A control (well-watered) was maintained for each genotype.Water was given on each pot (50% deficit and control) at 3-4 days intervals until seed set.Root and shoot samples were harvested on the 15th day after germination under control and drought-stressed plants.The entire experiment was conducted inside the greenhouse of the Biotechnology Department of ICAR-Umiam, maintained at 28 ± 2 °C temperature with a photoperiod of 16 h of light and 8 h of darkness.

Percentage of seed germination and ratio of shoot to root length
The percentage of seed germination was measured after one week of treatment with PEG.The percentage of germination was calculated as follows: Root and shoot length (cm) and root to shoot ratio were measured on 15th day after treatment.

Estimation of relative water content (RWC)
Relative water content (RWC) was estimated in the root and shoot tissues of control and PEG-treated rice genotype on 15th DAT as described by Baldoni et al. 61 .Briefly, Relative Water Content (RWC) was determined by weighing the fresh tissues (FW), followed by floating it on deionized water for 6 h under dim light at room temperature.Fully pompous tissues were re-weighed (PW), oven dried and again weight (DW) was measured.RWC was calculated using the formula: where FW Fresh weight, DW Dry weight and PW Pompous weight

Histochemical study using EVANs blue
Roots from the PEG-treated rice genotype were collected and stained with EVANs blue (Sigma-Aldrich) according to the method described by Vijayaraghavareddy et al. 62 .Briefly, 0.25 g of Evan's blue dye was dissolved in 100 ml of 0.1 M CaCl 2 solution at pH 5.6.Control and PEG-treated roots were excised and treated with EVANs blue for 30 min followed by rinsing with deionized water until the excess dye was removed.Qualitative estimation of EVANs staining performed under the light microscope.

Pollen fertility test
Five mature spikelets from three panicles each of Sahbhagi dhan, RCPL-1-82, RCPL-1-185 and IR64 were collected in the morning time (6-7 am) before anthesis.The collected samples were immediately fixed in FAA solution (formaldehyde: ethanol: acetic acid in the ratio of 1:18:1).Anthers were gently crushed, stained using I 2 -KI solution and observed under an optical microscope (LEICA).Circular dark-stained pollens were considered viable pollen, while irregular light-stained/destained pollens were considered infertile.

Estimation of total proline, hydrogen peroxide (H 2 O 2 ) and ascorbate content
Total Proline was extracted according to the method of Dien et al. 28 and estimated spectrophotometrically according to the method of Bates et al. 63 .Briefly, 100 mg root and shoot tissues were ground into fine powders using mortar and pestle.10 ml of 80% ethanol was mixed with a 100 mg powder sample and heated in the water bath at 80 °C for 45 min.The extracted materials were filtered and used for proline estimation. 1 ml of extract was mixed with 200 µl of acid ninhydrin and 500 200 µl of glacial acetic acid in a test tube.The mixer was boiled in the water bath for 45 min followed by being cooled in an ice bath for 5 min. 1 ml of toluene was added and the mixer was vortexed.The upper layer was measured at 520 nm using toluene as a blank.Proline content was determined using the proline (Sigma-Aldrich) standard curve at 520 nm and expressed as µmol g −1 DW.
H 2 O 2 concentration was measured colorimetrically as described by Jana and Choudhuri 64 .H 2 O 2 was extracted by homogenizing 100 mg tissues with 1 ml of phosphate buffer (pH 6.5) at low temperature followed by centrifugation at 6000×g for 25 min at 4 °C.The supernatant was taken in a fresh tube made up of 1 ml with phosphate buffer.H 2 O 2 content was estimated by mixing 500 µl extract with 500 µl of 0.1% titanium sulphate in 20% H 2 SO 4 (v/v) followed by centrifugation at 6000×g for 15 min at 4 °C.The yellowish colour intensity of the supernatant was measured at 410 nm using the H 2 O 2 (Sigma-Aldrich) standard curve.H 2 O 2 concentration was calculated using the extinction coefficient 0.28 mM −1 cm −1 and expressed as µmol g −1 tissue fresh weight.
Reduced ascorbate (AsA), dehydroascorbate (DHA), and total ascorbate (AsA + DHA) were extracted and quantified spectrophotometrically according to the method of Law et al. 65 .
The assay is based on AsA in an acidic solution reducing Fe3+ to Fe2+.The Fe2+ then combines with bipyridyl to produce a complex that absorbs at 525 nm and is coloured pink.The conversion of DHAs to AsA by dithiothreitol yields the measurement of total ascorbate (AsA + DHAs).100 µl sample extract was mixed with 200 µl of 10% (w/v) trichloroacetic acid followed by standing in the ice bath for 5 min.After adding 10 µl NaOH (1 M), the mixture was centrifuged for two minutes in a Microfuge.

Antioxidant enzymes assay
SOD was extracted by homogenizing 500 mg of fresh root and shoot samples in 5 ml of a buffer solution containing 100 mM potassium phosphate buffer (pH 7.5), 1.0 mM EDTA, 0.1 mM Triton X-100, and 2% polyvinylpyrrolidone (PVP).After centrifugation at 22,000×g for 10 min at 4 °C, the supernatant was dialyzed in cellophane membrane tubes against the cold extraction buffer.SOD was then measured by the method of Mishra and Fridovich 66 by observing epinephrine-dependent adrenochrome formation at 475 nm in a UV-Vis spectrophotometer.The Extraction media used for GR, DHAR and MDHAR were similar to SOD.GR activity assay was estimated using the method described by Foyer and Halliwell 10 using a reaction mixture of 80 mM Tris-HCl buffer (pH 8.5), 2.5 mM oxidized glutathione (GSSG), 1.5 mM EDTA, and 100 µl enzyme extract.The specific activity of the enzyme is expressed as nmol NADPH oxidised min −1 mg −1 protein.DHAR activity was determined according to the method described by Doulis et al. 11 using a reaction mixture containing 90 mM sodium potassium phosphate buffer (pH 7.0), 5.0 mM reduced glutathione (GSH), 0.1 mM EDTA and 100 µl enzyme extract.The enzyme-specific activity was expressed as nmol dehydroascorbate reduced min −1 mg −1 protein.MDHAR activity was estimated by the method of Hossain et al. 12 using a reaction mixture of 90 mM potassium phosphate buffer (pH 7.5), 0.2 mM NADH, 0.01 mM EDTA, 0.25 U ascorbate oxidase (Sigma), 0.0125% Triton X-100 and 100 µl enzyme extract.The MDHAR-specific activity was expressed as nmol NADH oxidised min −1 mg −1 protein.
CAT activity was estimated according to the method of Aebi 13 using 50 mM Tris-NaOH buffer.The rate of H 2 O 2 degradation was determined at 240 nm with an extinction coefficient of 0.036 mM −1 cm −1 .The specific activity of the enzyme was calculated as µmol H 2 O 2 oxidised min −1 mg −1 protein.GPX was extracted in 5 ml of cold 50 mM Na-phosphate buffer (pH 7.0) and measured according to the method given by Egley et al. 14 .Specific enzyme activity was expressed as µmol H 2 O 2 oxidized min −1 mg −1 protein.

DNA isolation, PCR amplification and genetic data analysis
Total genomic DNA was extracted from young leaves by the CTAB (cethyltrimethylammonium bromide, Himedia) method.To evaluate the degree of genetic diversity among 112 rice accessions, a total of 62 (57 SSR and 5 RAPD) molecular markers encompassing all 12 chromosomes were utilized.These markers were specific for drought-linked QTLs (Supplementary Table 2).SSR amplification reactions were prepared for a 25 µl reaction mixture containing 2 µl of DNA (20 ng), 1 unit of Taq DNA polymerase enzyme (Promega), 2 µl of 10× buffer, 2 µl of MgCl 2 (25 mM), 2 µl of dNTPs (2.5 mM each), 1 µl of primers (forward and reverse each), and 14.8 µl of H 2 O. SSR amplifications were performed with an initial denaturation step at 94 °C for 5 min, followed by 45 cycles of denaturation at 94 °C for 30 s, a primer annealing step at 62 °C for 45 s, an extension at 72 °C for 2 min and a final extension at 72 °C for 5 min.RAPD amplification reactions were prepared for a 25 μl reaction mixture containing 20 ng μl −1 template DNA, 10× buffer (NH 4 ) 2 SO 4 , 2.5 mM MgCl 2 , 3.0 mM dNTPs, 0.25 μM primers, and 1 unit of Taq DNA polymerase.RAPD amplifications were performed with an initial denaturation step at 94 °C for 7 min and 30 cycles at 94 °C for 1 min, 35 °C for 1 min, and 72 °C for 2 min; the final elongation step was performed at 72 °C for 6 min.The annealing temperature varied depending on the melting temperature of each primer.Each reaction was repeated thrice.The reaction products were analyzed by electrophoresis on 1.4% agarose gels, stained with ethidium bromide, and photographed under a UV transilluminator using a digital camera with a UV filter adapter.Synthetic DNA ladder 100 bp was used as a molecular marker for the molecular weight of the bands.Each amplified band profile was defined by the presence or absence of bands at specific positions on the gel.Profiles were considered distinct if at least one polymorphic band was identified.Fragments were scored as 1 if present or 0 if absent.Allele frequency and PIC (polymorphism information content) were calculated using PowerMarker V3.25 software 67 .A model-based population structure analysis was performed using STRU CTU RE software 68 .A phylogenetic tree was generated using NTSYSpc 2.10 based on the similarity index SM coefficient using 100,00 burn-in period length and 100,000 Markov Chain Monte Carlo (MCMC) repeats after burn-in 69 .

RNA isolation, cDNA synthesis and qRT-PCR
Total RNA was isolated from the root, shoot and young panicle tissues of control and treated rice genotype using TRIzol™ RNA purification kit (Thermofisher scientific) as per manufacturer's protocol.cDNA was synthesized using iScript cDNA synthesis kit (Biorad) following manufacturer's protocol.Identification of target drought-responsive genes was performed based on the available literature.Transcript abundance of DREB1 (Dehydration responsive element binding), p5cs (Pyrroline-5-carboxylate synthase), WRKY11, WRKY114, rcbs (Rubisco containing bodies), AAO1 (Aldehyde oxidase 1), SOD (Superoxide dismutase), SDR1, NAC9, ZFP252, ZFP182 and DRAP1 (Dr1-associated polypeptide-1), LOC_Os12g04500, LOC_Os02g50970, LOC_Os12g26290, LOC_Os05g08480, MYB80 were performed using quantitative real time PCR (qRT-PCR) and calculated as ΔCt (2 −ΔΔCT ) method 70 .The oligonucleotide primer pairs used for the amplification of drought-respective genes were designed using Primer3Plus software based on the gene sequence retrieved from NCBI and Rice Genome Database (http:// rice.plant biolo gy.msu.edu/ cgi-bin/ gbrow se/ rice/).Rice Actin gene was used to normalize the expression of drought responsive genes.A list of oligonucleotide sequences of these primers is shown in Supplementary Table 6.

Figure 2 .
Figure 2. A representative electrophoresis gel pic showing amplified DNA bands in 19 rice genotype using RAPD maker (a) and SSR marker (b); barplot (c) of 112 rice genotype developed based on the presence or absence of bands (SSR and RAPD markers) using STRU CTU RE software, similar colour indicates genetic similarity between the rice genotype; A line graph depicting K value of the population study (d).

Figure 3 .
Figure 3. Graphical representation of MDHAR, CAT, DHAR, GPX, GR and SOD activity in the shoot and root tissues of Sahbhagi Dhan, RCPL-1-82, RCPL-1-185 and IR-64 under PEG-mediated drought stress on 15th day after treatment.Each value is representation of mean ± sd, N = 9 and different letters indicates statically significant at p < 0.05.